The initiator methionine tRNA genes previously isolated from a recombinant phage library of human fetal liver DNA have been studied in greater detail. The functional states of the genes have been assayed in a newly described in vitro transcription system and in vivo, by insertion into a recombinant SV40 chromosome. Our studies have demonstrated that a variant tRNA(i)(met) gene, containing a base substitution in a sequence highly conserved in both prokaryotes and eukaryotes is not processed efficiently to a mature species in vitro and in vivo.